| In China,the death rate and mortality rate of mushroom poisoning ranks first in food poisoning.According to the National Public Health Incident Report Management Information System of the Chinese Center for Disease Control and Prevention,a total of 3701 mushroom poisoning cases were reported in China from 2004 to 2014,with 786 deaths and a mortality rate of 21.24%[3].In mushroom poisoning,about 90%of deaths are caused by mushrooms containing amatoxins[4].According to the composition and structure,toxins are classified into three types:amatoxins,phallotoxins and virotoxins.The most important lethal toxin is amatoxins[5].However,at present,the research on many important fields of amatoxins is not enough,which has caused no controversy so far.The main reasons are as follows:(1)Amatoxins is found in mushrooms of multiple genera,and each mushroom contains different toxins except amatoxins.It is difficult to describe the clinical diagnosis;(2)Insufficient research on toxicology mechanism leads to ambiguous target selection.The blindly searching for therapeutic drugs makes it difficult to achieve good therapeutic effects;(3)The lack of biomarkers for early diagnosis derived from the mechanism and pathway of poisoning makes it impossible to carry out rapid detection of Amanita toxin poisoning in clinical practice.The commonly used indexes of liver injury include alt,AST,TBIL and so on,which are often abnormal in the middle and late stage of the course of disease and have limited significance for early intervention.In view of this,this study mainly carried out work from the following three aspects:(1)Based on the literature review and meta analysis,the treatment measures of amanitin poisoning were classified according to the literature,and the different clinical treatment methods and effects of amanitin poisoning patients were systematically evaluated.The systematic literature review and quantitative analysis based on cross-sectional study can provide evidence support for the selection of treatment methods after amanitin poisoning;(2)To construct the animal and cell models of amanitin poisoning,and to explore the role of apoptosis,especially early apoptosis,in the liver injury induced by amanitin,and to study the change process of related upstream and downstream key proteins,so as to further supplement and improve the toxic chain of amanitin in vivo;(3)Apply metabolomics technology to analyze the liver's full metabolic profile of amanita poisoned mice,screen and analyze the different apparent changes of the body at different time periods after poisoning to find out the differential metabolites and related enrichment Pathway,to provide clinically possible biomarkers of amanita toxin poisoning effect,with a view to achieving early diagnosis and early intervention,and reducing the toxicity of amanita toxins Mortality mushroom poisoningPart ? Quantitative Evaluation of the Therapeutic Effect of Amatoxins Poisoning by Different Clinical Therapies Based on Meta-AnalysisObjective:Accidental ingestion of mushrooms containing amatoxin had resulted in many deaths worldwide in recent years,however,the clinical treatments for amatoxin poisoning were uniform and ineffective due to the strongest toxicity.Therefore,we sought to perform a retrospective Meta-analysis to evaluate the effect of different treatments strategies,and determine variations in time-and geography-dependent mortality rates after treatments in this study.Methods:PubMed,Embase,OVID,Wanfang and China National Knowledge Infrastructure were searched for qualified studies.Bias evaluation and sensitivity analysis were conducted for ensuring the quality of the results.Data manipulation and analyses were done using R softwareResults:2659 studies were collected,and 70 studies were finally included in the quantitative study.The model results showed that the overall mortality rate of amatoxin poisoning was 16.9%after various treatments,and 26.3%of ?-amatoxin poisoning.The mortality rate after co-treatment of detoxification,supportive measures and drugs showed the best(15.1%,with a 95%CI of 9.8-22.5%),which was 8.3%and 1.7%lower than that after a single treatment and dual treatments,respectively.The subgroup analysis indicated that patients in Asia were most vulnerable to amatoxin poisoning while treatment in Europe was more effective.Conclusion:Amatoxins poisoning has a high fatality rate and poor treatment effects,and there are significant differences between continents.The combination of drugs,detoxification and supportive therapy is the best.Part ? The ROS/p53-mediated mitochondrial apoptosis pathway is an early event of liver injury caused by ?-amanitinObjective:?-amanitin is considered to be the most important component of amatoxins.This part mainly discusses the role of apoptosis in ?-amanitin-induced liver injury and upstream activation signals..Methods:In animal experiments,BALB/C mice were randomly divided into three dose groups(0,0.23,0.35 mg/kg),with 6 mice in each group.We first observed the overall death and injury of ?-amanitin poisoning mice at different time periods(4,8,12,24,48,and 72 h)by TdT-mediated dUTP gap end labeling(TUNEL)method.We then further evaluated the levels of oxidase and antioxidant enzymes(SOD,MDA,and CAT)and the levels of proteins involved in apoptosis-related pathways(Cytochrome C,Caspase 8,Caspase 9 and Caspase 3)in mice liver tissue.In our in vitro model,L-02 cells are seeded in 96-or 6-well culture plates for detection of different indicators.Toxicity tests were performed at different time points(6,12,24,and 36 hours)with gradient concentrations(0,2 and 5 ?M)of ?-amanitin.The phase contrast microscope,cell counting kit(CCK-8)and flow cytometry were used to observe and detect the death and apoptosis of L-02 cells.Western Blotting was used to detect the expression levels of p53 protein and proteins related to the mitochondrial apoptotic pathway downstream(Bax,Bcl-2,Cytochrome C,Cleaved-Caspase 9 and Cleaved-Caspase 3).Then,we examined the relationship between oxidative damage and ?-amanitin-induced apoptosis of hepatocytes by measuring intercellular total ROS and mitochondrial reactive oxygen levels.Finally,we use the standard antioxidant N-acetyl-L-cysteine(NAC)to laterally verify the role of ROS in?-amanitin-induced L-02 cytotoxicity.Results1.?-amanitin causes damage to mouse liver tissue and L-02 cells In the first stage,the animal and cell models were constructed.In addition,with the increase of ?-amanitin concentration and/or the prolongation of treatment time,the growth of hepatocytes was significantly inhibited,and the growth density of hepatocytes under the microscope decreased significantly,and finally died..2.a-amanitin induces hepatocyte apoptosis through mitochondrial apoptotic pathwayNext,we discovered that ?-amanitin can cause liver damage and induce apoptosis,and that ?-amanitin-induced apoptosis may occur mainly through the mitochondrial apoptotic pathway.This is mainly manifested in the increase in the number of apoptotic cells in liver tissue(TUNEL staining)and L-02 cells(FITC Annexin V+7-AAD Apoptosis Detection Kit)after exposure to ?-amanitin.We also tested the levels of apoptosis-related proteins at the cell and animal levels and found that?-amanitin significantly altered the levels of p53 protein and downstream related proteins in the mitochondrial apoptotic pathway.Eventually,the mitochondrial apoptotic pathway may be locked as the main pathway.3.Oxidative stress may be an early event of hepatocyte apoptosis induced by?-amanitinIn the final stage of the experiment,we found that the average activity of SOD and CAT gradually decreased,and the average activity of oxidative stress products,especially MDA,increased significantly with the prolongation of the exposure time of?-amanitin.Moreover,the total ROS and mitochondrial ROS of exposed cells were significantly increased by flow cytometry analysis,suggesting that oxidative damage plays an important role in poisoned organisms.The reversal of poisoning after adding ROS inhibitors also confirms this conclusionConclusion:?-amanitin can affect the apoptosis process,including the expression of p53,Bax and Bcl-2,thus activating caspase cascade reaction related to mitochondrial apoptosis pathway.ROS,as an upstream signal molecule,participates in apoptosis induced by ?-amanitin.Part ? Energy disturbances caused by mitochondrial dysfunction are the main causes of liver damage and even liver failure caused by?-amanitinObjective:As one of the most important metabolic organs in the body,liver damage can cause systemic metabolic changes.Therefore,when studying the mechanism of liver damage,one should not only focus on the liver cells themselves,but also understand the changes of various metabolites in the body during the occurrence and development of diseases.The metabonomics research of ?-amanitin poisoning has not been reported yet,so the metabonomics technology is used in this study to analyze the whole metabonomics of the liver of mice exposed to ?-amanitin,to find out the different metabolites and their enrichment pathway,to find out the possible injury pathway and the time trend of injury from point to surface,and verify through molecular biology experiments..Methods:In this study,we screened and verified the possible pathway of liver injury and even liver failure caused by ?-amanitin from two aspects:in vivo animal and in vitro cell.Healthy BALB/c mice were randomly divided into 14 groups(0.23,0.35mg/kg dose group and 0,4,8,12,24,48,72h dose group,6 in each group).In the treatment group,0.5ml/kg ?-amanitin(0.23mg/kg and 0.35mg/kg,?-amanitin dissolved in normal saline)was injected intraperitoneally.The control group was given the same amount of normal saline(0.5ml/kg).1.After the successful establishment of the cell and animal model by liver enzyme index and cell survival rate,the inflammatory infiltration(including NF-?B p65,phosphorylated NF-?B p65,IKB ? and phosphorylated IKB ?)in the process of liver injury was observed by immunohistochemistry and Western blotting,and the main death mechanism of the cell was analyzed by flow cytometry with apoptosis and necrosis kit.2.To establish a Metabolomics model of mice poisoned by ?-amanitin based on gas chromatography-time-of-flight mass spectrometer(GC-TOF/MS),and to understand the effect of ?-amanitin on liver Combined with single factor and multivariate statistical analysis methods,and compared with the professional standard database,the different Metabolites were screened and identified.Finally,the target Metabolic pathway was identified through pathway enrichment.According to the relevant clues provided by GC-TOF/MS results,combined with molecular biology technology,the differential Metabolic pathway was verified and further studied.Results:1.hepatocyte necrosis and secondary reaction may be the important cause of death caused by ?-amanitinLike the results of the previous part of the experiment,the exposed mice showed obvious poisoning,and the exposed cells showed obvious decrease in survival rate.At the same time,according to the results of flow cytometry,the cells were mainly in the late stage of necrosis or apoptosis.Compared with the control group,the necrosis rate of cells in the exposed group increased significantly from 24 hours.According to the immunohistochemical evaluation,it was obvious that the positive staining of hepatocytes in the ?-amanitin exposed group increased significantly due to the activation of NF-?B(P<0.05),and showed a time-dependent trend compared with the control group.Similarly,the phosphorylation level of NF-? B and downstream protein I?B-? in ?-amanitin exposed group increased significantly with time compared with the control group(P<0.05).2.The effect of ?-amanitin on liver Metabolism2.1 general situation analysisThe overall analysis of all groups was carried out by principal components analysis(PCA)and partial least squares discrimination analysis,PLS-DA)model,the results showed that the changes of small molecule Metabolites in the liver of early mice were mainly concentrated in 8 h and 12 h after exposure to ?-amanitin,but after 48 h exposure,the liver of mice seemed to undergo the second stage of changes.2.2 analysis of potential biomarkers and related networks in liver of mice exposed to ?-amanitin1)A method of GC-TOF/MS was established to detect the Metabolic spectrum of mice model exposed to ?-amanitin.Through multivariate statistical analysis,it was found that there were significant differences in Metabolism between the exposed and non-exposed groups at 8h,12h and 48h2)During the period of 8h-12h after ?-amanitin exposure,the liver of mice changed in the first stage.Through the analysis of Metabolic pathway enrichment(mpea),we found that the Metabolites of starch and sucrose Metabolism,pantothenic acid and coenzyme A biosynthesis,and arginine biosynthesis changed significantly.D-glucose,d-maltose,glucose 6-phosphate,isomaltose,trehalose,?-ketoglutarate,ornithine,?-alanine,pantothenic acid,L-aspartic acid,L-cysteine,fumaric acid and n-acetylglutamic acid were selected as the highly significant compounds involved in this pathway,and there were differences in relative abundance among groups.Compared with the control group,the contents of 13 Metabolites in the exposure group changed significantly(P<0.05).Then we observed the new changes of mice after 48 hours of exposure to ?-amatoxin.In addition to the starch and sucrose Metabolism and arginine biosynthesis mentioned above,the citric acid cycle(TCA cycle)and galactose Metabolism also changed significantly.Similarly,we screened new high abundance Metabolites in these two pathways,including citric acid,fumaric acid,isocitric acid,?-ketoglutarate,pyruvic acid and D-galactose(P<0.05 compared with the control group).3.Mitochondrial dysfunction is an important cause of hepatocyte necrosis induced by ?-amanitinThe results of Metabolomics and the exploration of cell death mechanism indicate that mitochondrial dysfunction may be the main cause of hepatocyte damage and failure caused by ?-amanitin,even the early cause.The possible approaches include:inhibition of respiratory chain leading to ATP depletion,mitochondrial oxidative stress,mitochondrial permeability transition(MPT)-induced hepatocyte necrosis,etc.Therefore,we measured the permeability of mitochondrial transition pore(MPTP),the level of adenosine triphosphate(ATP),the production of mitochondrial reactive oxygen species(mtROS),and quantified the mitochondrial membrane potential(??m)using JC-1,which can also reflect the openness of MPTP.According to the results,?-amanitin significantly reduced ??m,increased mitochondrial permeability,decreased ATP and increased mtROS.All the results were related to time,especially after 24 hours.Conclusion:The abnormal metabolism of liver in mice exposed to ?-amanitin suggests that the metabolism of starch and sucrose,pantothenic acid and coenzyme A biosynthesis,arginine biosynthesis and citric acid cycle(TCA cycle)have changed significantly.Mitochondrial dysfunction may be the main cause of hepatic metabolic disorder induced by ?-amanitin. |
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